We will develop tools for near-infrared (NIR) image-guided interventions through the design, creation and testing of nanoscale imaging probes. This proposed effort will be accomplished through three aims. Aim 1: the design, fabrication and characterization of a unique set of nanoscale NIR probes for imaging tumor cells. Aim 2: the implementation of components for target directing of nanoscale probes. Aim 3: the generation of spatial and temporal image-based maps of tumor cells using NIR microscopy. Aim 1 will comprise the creation of water-soluble NIR quantum dots (QDs) that emit in the wavelength range from 850 to 2200 nm. This will be accomplished through modification of commercially available colloidal lead-sulfide (PbS) QDs which are stabilized in organic solvents using oleic acid. Oleic acid will be replaced with specific thiolated molecules rendering the QDs hydrophobic. Next, amphiphilic block copolymers will be used to encapsulate QDs and the overall structure will be locked by partially cross-linking the outer block. Characterization will be achieved through electron microscopy together with absorbance and fluorescence spectroscopy. Through Aim 2, the water soluble QDs will then be further modified through conjugation to a single chain fragment variable antibody (A33scFv) that recognizes the A33 surface protein which is expressed in 95% of primary and metastatic colorectal cancers. A covalent conjugation method will be optimized once the QD capping chemistry has been resolved. The specificity and sensitivity of the A33scFv-QD conjugates will be investigated through Aim 3 using the human colon carcinoma cell line SW1222 with control experiments using the A33-nonexpressing human colon cancer cell line HT29. The cells will be grown in glass-bottom Petri dishes and subsequently incubated with various concentrations of ScFv-QD conjugates. After various incubation times, cells will be washed and visualized under using an Olymus-BX51 fluorescence microscope equipped with both a CCD camera (bright-field imaging) and a NIR detector. Overlaid pictures of bright field and NIR images will be collected and NIR image analysis will permit high resolution imaging of the respective CT antigens allowing for their precise positioning and localization over time. The current sets of image-guided tools largely depend on the visible light spectrum which is limited by its depth of penetration. NIR probes will extend penetration depths providing better markers for guiding the surgeon. Our efforts are in line with the goals of the National Institute of Biomedical Imaging and Bioengineering in accelerating the application of biomedical technologies through integrating the physical, engineering, and life sciences. The proposed study is also aligned with the mission of the National Cancer Institute in advancing therapeutic delivery and it's goal to preempt cancer through improvements in early detection and diagnostics. This initial work will provide the technical foundation for the further development of tools that will help clinicians not only diagnose but more importantly employ image-guided therapies.